Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
In order to enhance their nutritional value, seed crops have been targets of genetic engineering efforts to either produce valuable proteins or alter the amino acid composition of existing proteins (Rademacher et al., 2009). However, what is frequently ignored is the subcellular function that proteins play in the development of the seed. In maize (Zea mays), the endosperm storage proteins constitute a major protein component in the seed. Most of them belong to the prolamins, common in many grass species, and in maize are referred to as zeins. The alcohol-soluble zein fraction extracted by the Osborne method without reducing agent is called zein-1 and consists mainly of the 19-1W (z1A, z1B, and z1D) and 22-1W (z1C)-zeins (Song and Messing, 2003). The fraction of alcohol-soluble proteins extracted with a disulfide reducing agent (Moureaux and Landry, 1968; Paulis et al., 1969; Landry and Moureaux, 1970) is called zein-2 (Sodek and Wilson, 1971) and is composed of γ-, β-, and -zeins (Esen, 1987; Coleman and Larkins, 1998).
α-Zeins with 26 (19-1W) and 16 (22-1W) gene copies in maize inbred B73 constitute 60% to 70% of total zeins. γ-Zeins consist of the 50-, 27-, and 16-1W proteins, each encoded by a single gene in B73, and amount to about 20% to 25% of total zeins. The 27- and 16-1W γ-zein genes originated from a common progenitor by allotetraploidization and share high DNA sequence similarity (Xu and Messing, 2008). The 50-1W γ-zein gene has low similarity to the other two γ-zein genes and its protein is barely detectable by SDS-PAGE (Woo et al., 2001). The 15-kD β-zein protein is encoded by a single gene and its product makes up 5% to 10% of total zeins (Thompson and Larkins, 1994). The 18- and 10-kD δ-zein proteins are also each encoded by a single gene and make up less than 5% of total zeins (Wu et al., 2009). From an evolutionary point of view, the α- and δ-zeins arose more recently, while the γ- and β-zeins are older and conserved across different subfamilies of the Poaceae (Xu and Messing, 2009).
Zeins are specifically synthesized in the endosperm about 10 days after pollination (DAP) on polyribosomes of the rough endoplasmic reticulum (RER), and the proteins are subsequently translocated into the lumen of the RER, where they assemble into protein bodies (Wolf et al., 1967; Larkins and Dalby, 1975; Burr and Burr, 1976; Lending and Larkins, 1992). Typical protein bodies at 18 to 20 DAP are spherical, discrete, 1 to 2 μm in diameter, and have a highly ordered architecture. α-Zeins and δ-zeins are deposited in the center of the protein body, while γ- and β-zeins are located in the peripheral layer (Ludevid et al., 1984; Lending and Larkins, 1989). Disturbance of the correct arrangement of zeins can result in irregular protein body shapes and opaque seed phenotypes (Coleman et al., 1997; Gillikin et al., 1997; Kim et al., 2004, 2006). However, the role of depletion of each class of zeins on the elaboration of protein bodies has not been studied because of the lack of natural mutants. Moreover, most existing opaque and floury mutants of maize have pleiotropic effects, which interfere with the determination of the role of storage proteins themselves.
Quality protein maize (QPM) is a high lysine-containing corn that is based on genetic modification of the opaque2 (o2) mutant. The O2 gene encodes a transcriptional activator of a subset of α-zein genes. Reduced levels of these proteins are compensated by the increased levels of lysine-rich proteins, thereby increasing the levels of lysine in the maize kernel. However, the non-vitreous phenotype of o2 makes the kernel soft, preventing commercial application because corn has to be stored in elevators and transported in large ship containers. Non-vitreous seeds are fragile and more vulnerable to fungal infection. In QPM, modifier genes convert the starchy endosperm of o2 to the vitreous phenotype of normal maize. There are multiple, unlinked o2 modifier loci (Opm) in QPM and their nature and mode of action are unknown. For conversion of elite lines into QPM, breeders first have to make both parental lines, used in hybrid seed production, homozygous for o2, and then convert them into QPM, respectively. During this process, breeders have to monitor the recessiveness of o2 and the presence of Mot, a lengthy process that discourages the spread of the benefits of QPM to consumers.
Although QPM breeding represents an advance compared to normal corn, resulting corn strains still exhibit relatively low protein content when compared to soybeans. Typical yellow dent maize contains 10% protein (Flint-Garcia et al, 2009), of which the essential amino acid lysine is only around 2% (Mertz et al, 1964), whereas soybean has 35% protein with sufficient levels of lysine. Therefore, maize meal is always supplemented with soybean in feed to meet the protein and lysine needs of livestock. However, soybean production is four times more expensive than corn. To take advantage of the cost difference, a well-known long-term selection-experiment was initiated in 1896 by C. G. Hopkins at the University of Illinois (Hopkins, 1899) and has lasted for more than a century (Dudley, 2007; Dudley & Lambert, 2004; Moose et al, 2004), yielding four strains, Illinois High Protein (IHP), Low protein (ILP), Reverse High protein (IRHP) and Reverse Low Protein (IRLP). Introgressed QTLs are capable of raising the protein concentration in IHP more than twice that in normal maize, with the most increased fraction being the alcohol-soluble proteins or prolamins. However, because the bulk of the protein consists of the lysine-poor prolamins, IHP corn could also not be commercialized. Despite all these efforts with QPM and IHP, it seems that breeders could not combine all three traits, high-lysine, hard endosperm, and high protein. Clearly a need exists in the art for improved methods to select for high-lysine, hard endosperm, and high protein and accelerate the introgression of these traits into any local germplasm.